ATOMIC FORCE MICROSCOPY FOR ANALYSIS OF AMYLOIDOGENESIS

（改编自本申请）淀粉样蛋白原纤维的形态具有 传统上通过电子显微镜进行分析。但这种方法 需要大量的样品制备和染色，例如， 乙酸双氧铀，这可能会影响形态。原子力显微镜（AFM） 允许在可能密切相关的条件下进行样品分析 生理条件。此外，AFM允许阐明 淀粉样蛋白生成途径，因为图像可以快速获得， 打断了这个过程。最新的AD阐明方法 淀粉样蛋白生成假设A β蛋白的给定变体形成淀粉样蛋白。 单纤维种。然而，大量证据表明， 阿尔茨海默氏病中淀粉样纤维的形态有细微的变化。这些 变异可能具有重要的生物学后果， 例如，它们与神经元表面的相互作用。因此有 重要的是阐明这些形态学差异，并确定 蛋白质聚集途径的动力学细节， 形态学原子力显微镜非常适合这项任务，因为与电子 显微镜，样品制备是最小的，实验可以 在生理相关条件下进行。申请人 建议利用AFM（1）来确定 淀粉样蛋白纤维从不同的前体和途径下获得 生理相关条件，（2）阐明体外生长 淀粉样蛋白的动力学，并评估内源性脑蛋白和小 从工业合作者获得的分子药物候选物影响 这些动力学，（3）直接探测小分子的结合能， 分子到淀粉样纤维，和（4）表征淀粉样纤维 以及死后脑组织中的弥漫性淀粉样蛋白的结果予以 研究将提供一个详细的了解机制，在体外 淀粉样蛋白的形成在纳米尺度上，而且， 评估小分子候选药物的作用和来源， 抑制淀粉样蛋白生成和体外数据与淀粉样蛋白的相关性 在体内形成。

(Adapted from the application) The morphology of an amyloid fibril has traditionally been analyzed by electron microscopy. However, this method requires extensive sample preparation and staining with, for example, uranyl acetate, which may affect morphology. Atomic force microscopy (AFM) allows sample analysis under conditions which may closely relevant physiological conditions. In addition, AFM allows the elucidation of the pathway of amyloidogenesis, since images can be obtained rapidly, without interrupting the process. Most current approaches to elucidation of AD amyloidogenesis assume that a given variant of the A beta protein forms a single fibrillar species. However, considerable evidence suggests that amyloid fibril morphology in Alzheimer's disease is subtly varied. These variations may have important biological consequence, affecting, for example, their interactions with neuronal surfaces. Therefore, it is important to elucidate these morphological differences and to determine the kinetic details of the protein aggregation pathways which lead to each morphology. AFM is perfectly suited for this task since, unlike electron microscopy, sample preparation is minimal and experiments can be conducted under physiologically-relevant conditions. The applicants propose to utilize AFM (1) to determine the structural morphology of amyloid fibers obtained from different precursors and pathways under physiologically relevant conditions, (2) to elucidate the in vitro growth kinetics of amyloid and to assess how endogenous brain proteins and small molecule drug candidates obtained from industrial collaborators affect these kinetics, (3) to probe directly the binding energetics of small molecules to amyloid fibers, and (4) to characterize the amyloid fibers and diffuse amyloid from post-mortem brain tissue. The results of these studies will provide a detailed understanding of the mechanism of in vitro amyloid formation at the nanometer length scale, and furthermore, will assess the effects and origin of small molecule drug candidates in inhibiting amyloidogenesis and the relevance of in vitro data to amyloid formed in vivo.